The ability to site-specifically incorporate unnatural amino acids (UAAs) into a protein in living cells has emerged as a powerful method to probe and manipulate its structure and function. Central to this technology is an engineered tRNA/aminoacyl-tRNA synthetase (aaRS) pair that delivers a desired UAA in response to a nonsense or frameshift codon. Such UAA-specific tRNA/aaRS pair must not cross-react with its host counterparts (i.e., orthogonal) to maintain the fidelity of translation. To ensure the absence of such cross-reactivity, candidates for the development of UAA-specific orthogonal tRNA/aaRS pairs are imported into a host cell from a different domain of life harboring evolutionarily divergent translational components. Thus, genetic code expansion of bacteria relies upon tRNA/aaRS pairs of eukaryotic or archaeal origin, and the same in eukaryotic cell generally utilizes bacterial pairs (homology of archaeal tRNA/aaRS pairs to their eukaryotic counterparts generally precludes their use in eukaryotic cells). The use of two distinct sets of tRNA/aaRS pairs for genetic code expansion in eukaryotes and bacteria has led to a significant disadvantage: each desirable UAA must be separately genetically encoded using two separate platforms.
The archaebacteria derived pyrrolysyl (Pyl) tRNA/PylRS pair is a natural TAG suppressor, and is orthogonal in both bacteria and eukaryotes owing to its unique structural features. As a result, its adaptation for genetic code expansion has created a universal platform that can be used to incorporate UAAs into proteins expressed in both E. coli and eukaryotic cells. The universal pyrrolysyl platform has been particularly beneficial for eukaryotic genetic code expansion for the following reason. Two selection systems have been developed so far to enable the generation of UAA-specific aaRS variants that use E. coli or Saccharomyces cerevisiae (yeast) as selection hosts to enable the directed evolution of eukaryotic-archaeal or bacterial tRNA/aaRS pairs, respectively. Due to its facile nature, the E. coli based selection platform has been significantly more successful for genetically encoding new UAAs relative to its yeast counterpart. The Pyl-tRNA/PylRS pair offers a unique opportunity to genetically encode new UAAs into eukaryotic cells using the facile E. coli based selection system. The advantage of this strategy is evident from the fact that all new UAAs genetically encoded in eukaryotic cells in the last six years have utilized the Pyl-tRNA/PylRS pair.
Development of additional “universal” tRNA/aaRS pairs that share these unique advantages, but provide access to new active site topologies for genetically encoding structurally distinct UAAs inaccessible to the pyrrolysyl system, would significantly augment our ability to expand and diversify the UAA tool box that can be used both in bacterial and eukaryotic cells. Access to multiple mutually orthogonal tRNA/aaRS pairs—each of which enable the incorporation of a rich set of UAAs—will also be crucial to facilitate site-specific incorporation of multiple distinct UAAs into proteins. Prolonged natural evolution has crafted the unique Pyl-tRNA/aaRS pair from a phenylalanyl ancestor a feat challenging to replicate in the laboratory setting.